Method for producing amino acids using bacterium of the enterobacteriaceae family

ABSTRACT

A method for producing an L-amino acid is described, for example, L-phenylalanine and L-histidine, by fermentation using a bacterium of the Enterobacteriaceae family, wherein the bacterium has been modified by attaching a DNA fragment able to be transcribed encoding the peptide represented in SEQ ID NO: 2, or a variant thereof, particularly a portion of the ssrA gene, to the 3′-end of gene encoding for the bacterial enzyme, which influences on the L-amino acid biosynthesis, such as chorismate mutase/prephenate dehydrogenase or phosphoglucose isomerase.

This application claims priority under 35 U.S.C. §119 to Russian PatentApplication No. 2007135818 filed on Sep. 27, 2007. This document isincorporated in its entirety by reference. The Sequence Listing inelectronic format filed herewith is also hereby incorporated byreference in its entirety (File Name: US-347_Seq_List; File Size: 34 KB;Date Created: Sep. 26, 2008).

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates to a method for producing an L-amino acidby fermentation, and more specifically to genes which aid in thisfermentation. These genes are useful for improving L-amino acidproduction, particularly L-phenylalanine and L-histidine.

2. Background Art

Conventionally, L-amino acids are industrially produced by fermentationmethods utilizing strains of microorganisms obtained from naturalsources, or mutants thereof. Typically, the microorganisms are modifiedto enhance production yields of L-amino acids. Many techniques toenhance L-amino acid production yields have been reported, includingtransformation of microorganisms with recombinant DNA (see, for example,U.S. Pat. No. 4,278,765). Other techniques for enhancing productionyields include increasing the activities of enzymes involved in aminoacid biosynthesis and/or desensitizing the target enzymes of thefeedback inhibition by the resulting L-amino acid (see, for example, WO95/16042 or U.S. Pat. Nos. 4,346,170, 5,661,012 and 6,040,160) andcreating bacterial strains which are deficient in the genes which usethe precursors of the target compound for other pathways, or creatingbacterial strains which are deficient in the genes responsible fordegradation of the target compound.

These manipulations usually result in strains which cannot grow, growonly at a significantly reduced rate, or require additional nutrients,such as amino acids. For example, enhancing the expression of some genesmay become excessive and could lead to significant inhibition ofbacterial growth and, as a result, lower the ability of the bacterium toproduce the target compound.

A possible approach for avoiding the difficulties described above is tooptimize the expression of the genes which encode proteins involved inthe distribution of the carbon, nitrogen, or phosphorus fluxes, or theexcretion of the target substance out of the bacterial cell.

This aim was often accomplished by introducing a mutation into apromoter sequence of the amino acid- or nucleic acid-biosynthesizinggenes (European Patent Application EP1033407A1), by obtaining thelibrary of synthetic promoters with different strengths (Jensen P. R.,and Hammer K., Appl. Environ. Microbiol., 1998, 64, No. 1. 82-87Biotechnol. Bioeng., 1998, 58, 2-3, 191-5), and creating a library ofartificial promoters (PCT application WO03089605).

It is known that tagging a protein with the ssrA peptide tag, encoded bySsrA RNA, marks the tagged protein for proteolytic degradation(Gottesmann S. and al, Genes & Dev., 12:1338-1347 (1998)). SsrA RNA(Synonyms: SsrA, tmRNA, 10Sa RNA, transfer-messenger RNA, SipB, B2621)functions to release a stalled ribosome from the end of a “broken” mRNAthat is missing a stop codon by acting both as a tRNA and as an mRNA,using a “trans-translation” mechanism. SsrA also mediates the release ofthe stalled ribosome due to lack of an appropriate tRNA (Hayes C. S. andal, PNAS, 99(6):3440-3445 (2002)). Other translation problems can alsolead to SsrA activity. In addition, SsrA RNA stimulates degradation ofdefective mRNAs (Yamamoto Y. et al, RNA, 9:408-418 (2003)).

The SsrA RNA contains a tRNA-like structural region that is processed byRNase P and charged by alanyl-tRNA synthetase (Komine Y et al, Proc.Natl. Acad. Sci. USA, 91 (20):9223-7 (1994)). The SsrA RNA also has aregion that acts as a messenger RNA and encodes a translated tag that isadded to the nascent protein and which targets this protein fordegradation (Tu G.-F. and al, J Biol Chem, 270(16):9322-9326 (1995)).The structure of the SsrA RNA and the translational mechanism have beenexamined in detail (Corvaisier S. et al, J Biol Chem, 278(17):14788-97(2003)). The SsrA RNA is transcribed as a larger precursor RNA that isthen processed to form the mature RNA.

SsrA is similar to RNAs from Mycoplasma capricolum, Bacillus subtilis(Muto A. and al, Genes Cells, 7(5):509-19 (2000)), Dichelobacternodosus, Synechococcus sp. strains PCC6301 and PCC6803, Thermusthermophilus, Salmonella enterica serovar Typhimurium, and to atwo-piece RNA from Caulobacter crescentus. The tmRNA genes have beenused as probes for the identification of bacterial species (SchonhuberW. and al, BMC Microbiology, 1(1):20 (2001)).

However, there have been no reports describing the use of ssrA-taggingfor L-amino acid production, for example, L-phenylalanine or L-histidineproduction. Particularly, there is no previous description of using abacterium of the Enterobacteriaceae family containing a DNA encoding thepeptide of SEQ ID NO: 2 or a variant thereof, attached immediatelydownstream of a gene encoding a bacterial enzyme which influencesL-amino acid biosynthesis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the scheme for tyrA gene extension.

FIG. 2 shows the relative positions of primers P1 and P2 on plasmidpMW118-attL-Cm-attR.

FIG. 3 shows the construction of the chromosomal DNA fragment with thedeleted pheA gene.

FIG. 4 shows the scheme for replacing the cat gene locus with the pheAgene.

FIG. 5 shows the alignment of the primary sequences of the tmRNA-encodedproteolysis-inducing peptide tag from Bacillus subtilis (BACSU),Bacillus stearothermophilus (BACST), Serratia marcescens (SERMA),Escherichia coli (ECOLI), Pseudomonas flurescens (PSEFL), Pseudomonaschlororaphis (PSECL), Pseudomonas putida (PSEUPU). The alignment wasdone by using the PIR Multiple Alignment program(http://pir.georgetown.edu). The identical amino acids are marked by anasterisk (*), similar amino acids are marked by a colon (:).

SUMMARY OF THE INVENTION

Aspects of the present invention include enhancing the productivity ofL-amino acid-producing strains and a method for producing L-amino acidsusing these strains. The above aspects were achieved by finding thatSsrA-tagging of an enzyme involved in the synthesis of a target aminoacid that shares common steps in its biosynthesis pathway with otheramino acids can enhance production of the target amino acid at theexpense of the amino acids having common biosynthesis steps and/orprecursors. It was also found that SsrA-tagging of an enzyme involved inthe distribution of the carbon fluxes between different pathways ofglycolysis (for example the pentose phosphate and Entner-Duodoroffpathways) can enhance production of those amino acids with precursorsproduced in one of the pathways of glycolysis. In either case, it wasfound that the prototrophic properties of the modified bacteria aremaintained.

This aim was achieved by constructing a new variant tyrA gene and a newvariant pgi gene, wherein the resulting proteins both have a C-terminalshort peptide sequence encoded by part of ssrA gene. It was shown thatthe use of this mutant TyrA-ssrA could enhance L-phenylalanineproduction when the mutant tyrA-ssrA gene is introduced into the cellsof the L-phenylalanine-producing strain in place of the native tyrAgene. It also was shown that the use of such mutant Pgi-ssrA couldenhance L-histidine production when the mutant pgi-ssrA gene isintroduced into the cells of the L-histidine-producing strain in placeof the native pgi gene. Thus, the present invention has been completed.

It is an aspect of the present invention to provide an L-amino acidproducing bacterium of the Enterobacteriaceae family comprising a DNAcomprising a gene encoding a bacterial enzyme which influences L-aminoacid biosynthesis, and a DNA fragment able to be transcribed andencoding the peptide of SEQ ID NO: 2, or a variant thereof, wherein saidDNA fragment is attached to said gene at the 3′ end of said gene.

It is a further aspect of the present invention to provide the bacteriumdescribed above, wherein said bacterium belongs to the genusEscherichia.

It is a further aspect of the present invention to provide the bacteriumdescribed above, wherein said bacterium is an L-phenylalanine producingbacterium.

It is a further aspect of the present invention to provide the bacteriumdescribed above, wherein the enzyme is chorismate mutase/prephenatedehydrogenase.

It is a further aspect of the present invention to provide the bacteriumdescribed above, wherein said bacterium is an L-histidine producingbacterium.

It is a further aspect of the present invention to provide the bacteriumdescribed above, wherein the enzyme is phosphoglucose isomerase.

It is a further aspect of the present invention to provide a method forproducing an L-amino acid comprising cultivating the bacterium describedabove in a culture medium, and isolating the L-amino acid from theculture medium.

It is a further aspect of the present invention to provide the methoddescribed above, wherein said L-amino acid is L-phenylalanine.

It is a further aspect of the present invention to provide the methoddescribed above, wherein said L-amino acid is L-histidine.

It is a further aspect of the present invention to provide a method forproducing a lower alkyl ester of α-L-aspartyl-L-phenylalanine,comprising cultivating the bacterium described above in a culture mediumto produce and accumulate L-phenylalanine in the medium, andsynthesizing the lower alkyl ester of α-L-aspartyl-L-phenylalanine fromthe aspartic acid or a derivative thereof and the obtainedL-phenylalanine.

It is a further aspect of the present invention to provide the methoddescribed above, wherein the method further comprising esterifyingL-phenylalanine to generate a lower alkyl ester of L-phenylalanine,condensing the lower alkyl ester of L-phenylalanine with the asparticacid derivative, wherein the derivative is N-acyl-L-aspartic anhydride,separating the lower alkyl ester of N-acyl-α-L-aspartyl-L-phenylalaninefrom the reaction mixture, and hydrogenating the lower alkyl ester ofN-acyl-α-L-aspartyl-L-phenylalanine to generate the lower alkyl ester ofα-L-aspartyl-L-phenylalanine.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 1. Bacterium of thePresent Invention

It is known that some L-amino acids have common precursors duringbiosynthesis by a cell, such as aromatic amino acids, branched chainamino acids, etc. When producing such an amino acid in a bacterialstrain, it is useful to make the strain auxotrophic for the other aminoacids which have a common precursor with the targeted amino acid. Thiscan prevent the common precursor from directing the biosynthesis awayfrom the biosynthetic pathway of the targeted amino acid.

Another method for ensuring production of the targeted amino acid, asopposed to those having a common precursor, is to generate a so called“leaky” mutation in an enzyme which is responsible for producing theother amino acids from the common precursor. In this case, it is notnecessary to feed the bacterium with the other L-amino acid(s), sincesynthesis of these L-amino acids in the bacterium is suppressed.

SsrA-tagging of an enzyme results in a reduction in the enzyme activity,since the enzyme is degraded by the transcribed ssrA-tagged RNA viaRNase P (Komine Y et al, Proc. Natl. Acad. Sci. USA, 91 (20):9223-7(1994)) and proteolysis of the ssrA-tagged enzyme by ClpXP and ClpAPproteases (Wah D. A. et al, Chem. Biol., 9(11):1237-45 (2002)). Suchmodification by ssrA-tagging permits retention of the prototrophicproperties of the modified bacterium, while exhibiting a leaky-typephenotype. Also, the ssrA-tagging permits the reduction of the level ofby-products by decreasing the activity of one or several enzymesinvolved in the biosyntesis of such by-products.

“L-amino acid-producing bacterium” means a bacterium which has anability to cause accumulation of an L-amino acid in a medium when thebacterium is cultured in the medium. The L-amino acid-producing abilitymay be imparted or enhanced by breeding. The phrase “L-aminoacid-producing bacterium” as used herein also means a bacterium which isable to produce and cause accumulation of an L-amino acid in a culturemedium in an amount larger than a wild-type or parental strain of E.coli, such as E. coli K-12, and preferably means that the microorganismis able to cause accumulation in a medium of an amount not less than 0.5g/L, more preferably not less than 1.0 g/L of the target L-amino acid.The term “L-amino acids” includes L-alanine, L-arginine, L-asparagine,L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, L-glycine,L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine,L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan,L-tyrosine, and L-valine. L-histidine is particularly preferred.Aromatic amino acid, such as L-phenylalanine, L-tryptophan andL-tyrosine is more preferred and L-phenylalanine is particularlypreferred.

The Enterobacteriaceae family includes bacteria belonging to the generaEscherichia, Enterobacter, Erwinia, Klebsiella, Pantoea, Providencia,Salmonella, Serratia, Shigella, Morganella, etc. Specifically, thoseclassified into the Enterobacteriaceae according to the taxonomy used inthe NCBI (National Center for Biotechnology Information) database(http://www.ncbi.nlm.nih.gov/htbinpost/Taxonomy/wgetorg?mode=Tree&id=1236&lvl=3&keep=1&srchmode=1&unlock)can be used. A bacterium belonging to the genus Escherichia or Pantoeais preferred.

The phrase “a bacterium belonging to the genus Escherichia” means thatthe bacterium is classified into the genus Escherichia according to theclassification known to a person skilled in the art of microbiology.Examples of a bacterium belonging to the genus Escherichia as used inthe present invention include, but are not limited to, Escherichia coli(E. coli).

The bacterium belonging to the genus Escherichia is not particularlylimited; however, for example, bacteria described by Neidhardt, F. C. etal. (Escherichia coli and Salmonella typhimurium, American Society forMicrobiology, Washington D.C., 1208, Table 1) are encompassed.

The bacterium encompasses a strain of the Enterobacteriaceae familywhich has an ability to produce an L-amino acid and has been modified byattaching a DNA fragment having part of the ssrA gene to the 3′-end of agene encoding a bacterial enzyme. The part of the ssrA gene encodes forthe peptide represented in SEQ ID NO: 2 or a variant thereof, and thebacterial enzyme is involved in the biosynthetic pathway of an L-aminoacid which has a common precursor with the targeted L-amino acid. Inaddition, the bacterium encompasses a strain of the Enterobacteriaceaefamily which has an ability to produce an L-amino acid and has beentransformed with a DNA fragment encoding part of the ssrA gene so thatcomponents of the peptide encoded by the DNA fragment are expressed.

The ssrA gene (synonyms: ECK2617, b2621, sipB) encodes the tmRNA(synonyms: SsrA, 10Sa RNA, transfer-messenger RNA, SipB, B2621). ThessrA gene (nucleotides from 2753615 to 2753977 in the sequence ofGenBank accession NC_(—)000913.2, gi: 49175990) is located between thesmpB and intA genes on the chromosome of E. coli K-12. The ssrA genefrom Escherichia coli is represented by SEQ ID NO: 1. The sequence ofthe tmRNA-encoded proteolysis-inducing peptide tag is represented by SEQID NO: 2. This peptide is encoded by nucleotides on positions from 90 to119 of gene ssrA represented in SEQ ID NO: 1. Other ssrA genes have alsobeen elucidated as follows: tmRNA-encoded proteolysis-inducing peptidetag from Bacillus subtilis, Bacillus stearothermophilus, Serratiamarcescens, Escherichia coli, Pseudomonas flurescens, Pseudomonaschlororaphis, and Pseudomonas putida.

Bacteria which are able to concurrently produce two L-amino acids whichhave a common precursor may be used. The goal of the present inventionwas achieved by using a strain which had been previously modified toconcurrently produce two amino acids, namely L-phenylalanine andL-tyrosine. Such a strain can be obtained by destroying thetranscriptional dual regulator, namely, the tyrR gene by the standardRed-driven recombination method. Deletion of the tyrR gene can beconfirmed by PCR using the chromosomal DNA of the transformed strain asa template. Bacteria which are able to produce L-amino acids may beused, and preferably bacteria which are able to concurrently producephenylalanine and tyrosine E. coliMG1655 ΔtyrR is an example of such astrain. E. coli strain MG1655 ΔtyrR was obtained by substitution of thechromosomal tyrR gene with a DNA cassette containing the Cm markerfollowed by excision of the marker using standard techniques describedin WO 05/010175. Also, an L-histidine producing strain in which the pgigene is modified by ssrA-tagging to increase flux ofD-glucose-6-phosphate to the pentose phosphate pathway to enhanceproductivity of L-histidine is also encompassed and described.

The phrase “a DNA fragment able to be transcribed and encoding thepeptide of SEQ ID NO: 2 or a variant thereof, wherein said fragment isattached to said gene at the 3′ end of said gene” means that the geneencoding the bacterial enzyme has been modified to have additionalnucleotide residues on its 3′-end as compared to a non-modified gene,for example, a gene of the wild-type strain. The presence of this DNAfragment (SEQ ID NO: 2 or a variant thereof) at the 3′-end of the geneencoding the bacterial enzyme leads to the formation of a tagged mRNAspecies, and the translated corresponding protein has an additionalpeptide tagged, or attached to, the C-terminus thereof. The bacterialenzyme may be modified by methods that include genetic recombining,C-terminal extensions, C-terminal fusions, and so forth. The length ofthe modified enzyme protein can be measured, for example, by Westernblot with specific antibodies, protein sequencing, and the like. Thephrase “attaching the DNA fragment encoding the peptide of SEQ ID NO: 2or a variant thereof to the carboxyl terminus of the protein” can alsomean that the translated modified protein is expressed in the bacteriumin such a way that the modified protein is degraded at a controllablerate (McGinness K. E. and al, Molecular Cell, 22: 701-707 (2006)).

The phrase “a DNA fragment able to be transcribed” means that the DNAfragment is attached to the 3′-end of the gene coding for the bacterialenzyme in such way that the stop-codon of the gene encoding thebacterial enzyme is removed and the necessary DNA fragment is introducedimmediately after the last codon of the gene. Such a DNA construct istranscribed as one transcriptional unit tagging the desired DNA fragmentto the mRNA of the gene of interest (the bacterial enzyme). It alsoresults in expression of ssrA-tagged proteins.

The phrase “a variant thereof” means a peptide which has changes in thesequence, whether they are deletions, insertions, additions, orsubstitutions of amino acids, but still maintains the desired activityat a useful level, for example, useful for decreasing the activity oftagged protein and consequently enhancing production of an L-amino acid.The number of changes in the variant peptide depends on the position orthe type of amino acid residues in the primary structure of the peptide.The number of changes may be 1 to 4 and preferably 1 to 2 for thepeptide listed as SEQ ID NO: 2. These changes in the variants can occurin regions of the peptide which are not critical for the function of thepeptide. This is because some amino acids have high homology to oneanother so the activity is not affected by such a change. Therefore, thepeptide variants may have a homology of not less than 70%, preferablynot less than 80%, and more preferably not less than 90%, and mostpreferably not less than 95% with respect to the entire amino acidsequences shown in SEQ ID NO. 2 as long as the ssrA-tagged protein isrecognized by proteases resulting in subsequent degradation. Homologybetween two amino acid sequences can be determined using the well-knownmethods, for example, the computer program BLAST 2.0, which calculatesthree parameters: score, identity and similarity.

The substitution, deletion, insertion, or addition of one or severalamino acid residues should be conservative mutation(s) so that theactivity is maintained. The representative conservative mutation is aconservative substitution. Examples of conservative substitutionsinclude substitution of Ser or Thr for Ala, substitution of Gln, His orLys for Arg, substitution of Glu, Gln, Lys, His or Asp for Asn,substitution of Asn, Glu or Gln for Asp, substitution of Ser or Ala forCys, substitution of Asn, Glu, Lys, His, Asp or Arg for Gln,substitution of Asn, Gln, Lys or Asp for Glu, substitution of Pro forGly, substitution of Asn, Lys, Gln, Arg or Tyr for His, substitution ofLeu, Met, Val or Phe for Ile, substitution of Ile, Met, Val or Phe forLeu, substitution of Asn, Glu, Gln, His or Arg for Lys, substitution ofIle, Leu, Val or Phe for Met, substitution of Trp, Tyr, Met, Ile or Leufor Phe, substitution of Thr or Ala for Ser, substitution of Ser or Alafor Thr, substitution of Phe or Tyr for Trp, substitution of His, Phe orTrp for Tyr, and substitution of Met, Ile or Leu for Val.

Data comparing the primary sequences of tmRNA-encodedproteolysis-inducing peptide tag from Bacillus subtilis (BACSU),Bacillus stearothermophilus (BACST), Serratia marcescens (SERMA),Escherichia coli (ECOLI), Pseudomonas flurescens (PSEFL), Pseudomonaschlororaphis (PSECL), Pseudomonas putida (PSEUPU) show a high level ofhomology of the amino acid sequence “YALAA” in the C-terminus (see FIG.5). It is possible to substitute similar (marked by colon) amino acidsresidues by the similar amino acid residues without deterioration of thepeptide activity. But modifications of other non-conserved amino acidresidues may not lead to alteration of the activity of tmRNA-encodedproteolysis-inducing peptide tag. Peptide tagged amino acid sequencesreferred to by McGinness K. E. et al. (Molecular Cell, 22:701-707(2006)) can also be considered as a peptide variant.

The DNAs which encode substantially the same peptides as components ofthe tag peptide may be obtained, for example, by modifying thenucleotide sequences of DNAs encoding components of tag peptide (SEQ IDNO: 1), for example, by means of the site-directed mutagenesis method sothat one or more amino acid residues at a specified site involvedeletion, substitution, insertion, or addition. DNAs modified asdescribed above may be obtained by conventionally known mutationtreatments. Such treatments include hydroxylamine treatment of the DNAencoding proteins, or treatment of the bacterium containing the DNA withUV irradiation or a reagent such as N-methyl-N′-nitro-N-nitrosoguanidineor nitrous acid. DNAs encoding substantially the same peptides as tagpeptide can be obtained by expressing DNAs having a mutation asdescribed above in an appropriate cell, and investigating the activityof the expressed product. DNAs encoding substantially the same peptideas tag peptide can also be obtained by isolating DNAs that arehybridizable with probes having nucleotide sequences which contain, forexample, the nucleotide sequences shown in SEQ ID NO: 1 under thestringent conditions, and encode peptides having the activities of tagpeptide. The “stringent conditions” referred to herein are conditionsunder which so-called specific hybrids are formed, and non-specifichybrids are not formed. For example, stringent conditions can beexemplified by conditions under which DNAs having high homology, forexample, DNAs having homology of not less than 50%, preferably not lessthan 60%, more preferably not less than 70%, further preferably not lessthan 80%, and still more preferably not less than 90%, and mostpreferably not less than 95% are able to hybridize with each other, butDNAs having homology lower than the above are not able to hybridize witheach other. Alternatively, stringent conditions may be exemplified byconditions under which DNA is able to hybridize at a salt concentrationequivalent to ordinary washing conditions in Southern hybridization,i.e., 1×SSC, 0.1% SDS, preferably 0.1×SSC, 0.1% SDS, at 60° C. Durationof washing depends on the type of membrane used for blotting and, as arule, what is recommended by the manufacturer. For example, recommendedduration of washing, for example, for the Hybond™ N+ nylon membrane(Amersham), under stringent conditions approximately is 15 minutes.Preferably, washing may be performed 2 to 3 times.

Partial sequences of the nucleotide sequence of SEQ ID NO: 1 can also beused as probes. Probes may be prepared by PCR using primers based on thenucleotide sequence of SEQ ID NO: 1 and DNA fragments containing thenucleotide sequence of SEQ ID NO: 1 as templates.

The substitution, deletion, insertion, or addition of nucleotides asdescribed above also includes mutations which naturally occur (mutant orvariant), for example, due to variety in the species or genus ofbacterium, which contains the components of tag peptide.

Expression of a bacterial gene which has a DNA fragment encoding thepeptide represented in SEQ ID NO: 2, or a variant thereof, attached tothe 3′-end of the gene can be achieved by transforming a bacterium withthe DNA encoding the peptide, and more exactly, by introducing the DNAfragment into the bacterial chromosome downstream of the gene encodingthe protein without disturbing the coding frame, or preferably, byreplacing the native chromosomal gene with a mutant gene encoding thetagged protein by conventional methods. Transformation of a bacteriumwith DNA will result in the formation of a new chromosomal mutant geneencoding the elongated protein. Methods of transformation include anyknown methods that have hitherto been reported.

For example, the following methods may be employed to introduce the DNAencoding the mutant gene by gene recombination. A mutant gene isprepared, and the bacterium is transformed with a DNA fragmentcontaining the mutant gene. Then, the native gene on the chromosome isreplaced with the mutant gene by homologous recombination, and theresulting strain is selected. Such gene replacement by homologousrecombination can be conducted by employing a linear DNA, which is knownas “Red-driven integration” (Datsenko, K. A. and Wanner, B. L., Proc.Natl. Acad. Sci. USA, 97, 12, p 6640-6645 (2000)), or by methodsemploying a plasmid containing a temperature-sensitive replication (U.S.Pat. No. 6,303,383 or JP 05-007491A). To increase the permeability ofthe cells to the DNA, treating the recipient cells with calcium chloridehas been reported for Escherichia coli K-12 (Mandel, M. and Higa, A., J.Mol. Biol., 53, 159 (1970)) and may be used.

Methods for preparation of plasmid DNA include, but are not limited to,digestion and ligation of DNA, transformation, selection of anoligonucleotide as a primer and the like, or other methods well known toone skilled in the art. These methods are described, for instance, inSambrook, J., Fritsch, E. F., and Maniatis, T., “Molecular Cloning ALaboratory Manual, Second Edition”, Cold Spring Harbor Laboratory Press(1989).

In the present invention, the phrase “bacterial enzyme which influencesL-amino acid biosynthesis” means an enzyme involved in the biosyntheticpathway of the undesired product, for example, the product which sharesa common precursor with the targeted L-amino acid. The phrase “bacterialenzyme involved in the biosynthetic pathway of the undesired product”means an enzyme catalyzing the reaction of a common precursor of thetargeted L-amino acid and the undesired product being converted into athe undesired product. This happens because the enzyme directs thereaction down the branch of the biosynthetic pathway which results inproduction of the undesired product. The phrase “bacterial enzyme whichinfluences L-amino acid biosynthesis” also means an enzyme involved inthe biosynthesis pathway of by-products of the targeted L-amino acid.The presence of such by-products can cause significant technicalproblems during purification, and negatively effect the L-amino acidproduction.

Genes encoding for the bacterial enzyme which influences L-amino acidbiosynthesis are the genes of interest. Such genes are represented by,but not limited to, tyrA gene encoding for chorismate mutase/prephenatedehydrogenase, pgi gene encoding for phosphoglucose isomerase, ilvE geneencoding for branched-chain amino-acid aminotransferase, ilvA and tdcBgenes encoding for threonine dehydratases, sdaA and sdaB genes encodingfor L-threonine deaminases, argA gene encoding for N-acetylglutamatesynthase, argG gene encoding for argininosuccinate synthase, proB geneencoding for γ-glutamyl kinase, thrB gene encoding for homoserinekinase, gene encoding for homoserine dehydrogenase etc. In general, anygene encoding an enzyme which causes the production of a metaboliteresulting from deviation from the pathway of the target L-amino acidand/or involved in the formation of a by-product is the gene ofinterest.

Chorismate mutase/prephenate dehydrogenase (synonyms: B2600, TyrA)catalyzes reversible conversion between chorismate and prephenate, andreversible conversion between prephenate and p-hydroxyphenylpyruvate,which is the intermediate compound in the pathway biosynthesis oftyrosine. Chorismate conversion is the first step in the biosynthesis ofboth tyrosine and phenylalanine. Modification of the TyrA protein byssrA-tagging can be useful for decreasing the flux of prephenate to theL-tyrosine biosynthetic pathway, and enhancing the production ofL-phenylalanine. The activity of chorismate mutase/prephenatedehydrogenase can be measured by using a stopped-time assay in thepresence of 2.5 mM chorismate or by immunodiffusion analysis of theenzyme-antibody complexes in plates prepared with 0.8% agarose (Rood J.I., Perrot B. et al., Eur J Biochem.; 124, 513-519 (1982)). Thewild-type tyrA gene (synonyms: ECK2597, b2600) which encodes thechorismate mutase/prephenate dehydrogenase from Escherichia coli hasbeen elucidated. The tyrA gene (nucleotides complementary to nucleotides2736970 to 2738091 in the sequence of GenBank accession NC_(—)000913.2,gi: 49175990) is located between the pheA and aroF genes on thechromosome of E. coli K-12. The tyrA gene from Escherichia coli isrepresented by SEQ ID NO: 3, and the amino acid sequence encoded by thetyrA gene is represented by SEQ ID NO: 4.

In the biosynthetic pathway of phenylalanine, reversible conversionbetween prephenate and phenylpyruvate is catalyzed by chorismatemutase/prephenate dehydratase (synonyms: B2599, PheA), which subunit isencoded by pheA gene (synonyms: ECK2596, b2599) in E. coli. The pheAgene (nucleotides from 2735767 to 2736927 in the sequence of GenBankaccession NC_(—)000913.2, gi: 49175990) is located between the pheA geneleader peptide and tyrA gene on the chromosome of E. coli K-12. The pheAgene from Escherichia coli is represented by SEQ ID NO: 5, and the aminoacid sequence encoded by the pheA gene is represented by SEQ ID NO: 6.Simultaneous modification of the TyrA and PheA proteins by ssrA-taggingcan be useful for decreasing the flux of chorismate to the L-tyrosineand L-tyrosine biosynthetic pathway and enhancing the production ofL-phenylalanine.

Phosphoglucose isomerase (synonyms: B4025, Pgi, glucose-6-phosphateisomerase, D-glucose-6-phosphate-ketol-isomerase) catalyzes reversibleconversion between β-D-glucose-6-phosphate and D-fructose-6-phosphate ingluconeogenesis, and glycolysis pathways. Decreasing phosphoglucoseisomerase activity leads to the redistribution of carbon fluxes in favorof the pentose phosphate pathway in which histidine precursor metabolite5-phosphoribosyl 1-pyrophosphate is generated. Activity ofphosphoglucose isomerase can be detected by, for example, the methoddescribed in Friedberg I. (J Bacteriol., 112, 3:1201-1205 (1972)) in acoupled assay by using G6P dehydrogenase (Winkler H. H., J Bacteriol.,101, 2:470-475 (1970)). The wild-type pgi gene (synonyms: ECK4017,b4025) which encodes the glucose-6-phosphate isomerase from Escherichiacoli has been elucidated. The pgi gene (nucleotides from 4,231,781 to4,233,430 in the sequence of GenBank accession NC_(—)000913.2, gi:49175990) is located between the lysC and yjbE genes on the chromosomeof E. coli K-12. The pgi gene from Escherichia coli is represented bySEQ ID NO: 7, and the amino acid sequence encoded by the pgi gene isrepresented by SEQ ID NO: 8.

Branched-chain amino acid aminotransferase (synonyms: B3770, IlvE)catalyzes a series of transamination reactions, each of which generatesα-ketoglutarate and one of three aliphatic branched chain amino acids.Modification of the IlvE protein by ssrA-tagging can be useful fordecreasing the flux of 2-ketoisovalerate to the L-valine biosyntheticpathway, which also results in decreasing the amount of L-isoleucine asby-product and enhancing the production of L-leucine. The activity ofbranched-chain amino acid aminotransferase can be measured by the methoddescribed, for example, in Lee-Peng F C et al (J. Bacteriol., 139(2);339-45 (1979)). The wild-type ilvE gene (synonyms: ECK3762, b3770,threonine deaminase, L-threonine hydrolyase) which encodes the branchedchain amino acid aminotransferase from Escherichia coli has beenelucidated. The ilvE gene (nucleotides 3,950,507 to 3,951,436 in thesequence of GenBank accession NC_(—)000913.2, gi: 49175990) is locatedbetween the ilvM and ilvD genes on the chromosome of E. coli K-12.

Threonine dehydratase (synonyms: B3772, Ile, IlvA) catalyzes thedeamination of L-threonine. Modification of the IlvA protein byssrA-tagging can be useful for preventing L-theonine degradation anddecreasing the flux of L-threonine to the L-isoleucine biosyntheticpathway, as well as being useful for L-arginine production. The activityof threonine dehydratase can be measured by the method described, forexample, in Eisenstein, E. (J. Biol. Chem., 266(9); 5801-7 (1991)). Thewild-type ilvA gene (synonyms: ECK3764, b3772, ile) which encodes thethreonine dehydratase from Escherichia coli has been elucidated. TheilvA gene (nucleotides 3,953,354 to 3,954,898 in the sequence of GenBankaccession NC_(—)000913.2, gi: 49175990) is located between the ilvD andilvY genes on the chromosome of E. coli K-12.

Threonine dehydratase (synonyms: B3117, Tdc, TdcB, threonine deaminase,L-serine dehydratase, serine deaminase, L-threonine hydrolyase)catalyzes deamination of L-threonine. Modification of the TdcB proteinby ssrA-tagging can be useful for preventing L-theonine degradation anddecreasing the flux of L-threonine to the L-isoleucine biosyntheticpathway. The wild-type tdcB gene (synonyms: ECK3106, b3117, tdc) whichencodes the threonine dehydratase from Escherichia coli has beenelucidated. The tdcB gene (nucleotides complementary to nucleotides3,263,061 to 3,264,050 in the sequence of GenBank accessionNC_(—)000913.2, gi: 49175990) is located between the tdcC and tdcA geneson the chromosome of E. coli K-12.

Threonine deaminases SdaA (synonyms: B1814, SdaA, SDH1, L-SD,L-threonine deaminase I, L-serine dehydratase 1, SDH-1, L-SD1,L-hydroxyaminoacid dehydratase 1, L-serine deaminase 1, L-serinehydro-lyase 1) and SdaB (synonyms: B2797, SdaB, L-threonine deaminaseII, L-serine deaminase 2, L-serine dehydratase 2, SDH-2, L-SD2, L-serinehydrolyase 2) catalyze deamination of L-threonine. Modification of thethreonine deaminase protein by ssrA-tagging can be useful for preventingL-theonine degradartion and decreasing the flux of L-threonine to theL-isoleucine biosynthetic pathway. The wild-type sdaA gene (synonyms:ECK1812, b1814) and sdaB gene (synonyms: ECK2792, b2797) which encodethe threonine deaminases from Escherichia coli have been elucidated. ThesdaA gene (nucleotides 1,894,956 to 1,896,320 in the sequence of GenBankaccession NC_(—)000913.2, gi: 49175990) is located between the yeaB andyoaD genes on the chromosome of E. coli K-12. The sdaB gene (nucleotides2,927,598 to 2,928,965 in the sequence of GenBank accessionNC_(—)000913.2, gi: 49175990) is located between the sdaC and xni geneson the chromosome of E. coli K-12.

N-acetylglutamate synthase (synonyms: B2818, ArgA, NAGS,acetyl-CoA:L-glutamate N-acetyltransferase,amino-acid-N-acetyltransferase) catalyzes the synthesis ofN-acetylglutamate from L-glutamate and acetyl-CoA, which is the firststep in the superpathway of arginine and ornithine biosynthesis.Modification of the ArgA protein by ssrA-tagging can be useful toaccumulate glutamate for L-proline production. The activity ofN-acetylglutamate synthase can be measured by the method described, forexample, in Marvil, D. K. and Leisinger, T. (J. Biol. Chem., 252(10);3295-303 (1977)). The wild-type argA gene (synonyms: ECK2814, Arg2,Arg1, b2818) which encodes the N-acetylglutamate synthase fromEscherichia coli has been elucidated. The argA gene (nucleotides2,947,264 to 2,948,595 in the sequence of GenBank accessionNC_(—)000913.2, gi: 49175990) is located between the amiC and recD geneson the chromosome of E. coli K-12.

Argininosuccinate synthase (synonyms: B3172, ArgG, argininosuccinatesynthetase, citrulline-aspartate ligase, L-citrulline:L-aspartateligase) catalyzes the synthesis of argininosuccinate L-aspartate andcitrulline. Modification of the ArgG protein by ssrA-tagging can beuseful for citrulline production. The wild-type argG gene (synonyms:ECK3161, b3172) which encodes the argininosuccinate synthase fromEscherichia coli has been elucidated. The argG gene (nucleotides3,316,659 to 3,318,002 in the sequence of GenBank accessionNC_(—)000913.2, gi: 49175990) is located between the argR and yhbX geneson the chromosome of E. coli K-12.

γ-glutamyl kinase (synonyms: B0242, ProB, glutamate 5-kinase, GK,ATP:L-glutamate 5-phosphotransferase, G5K) catalyzes the reaction ofglutamate phosphorylation, which is the first reaction in the synthesisof proline. Modification of the ProB protein by ssrA-tagging can beuseful to accumulate glutamate for L-arginine production. The activityof γ-glutamyl kinase can be measured by the method described, forexample, in Smith, C. J. et al, (J. Bacteriol., 157(2); 545-51 (1984)).The wild-type proB gene (synonyms: ECK0243, pro(2), b0242, pro2) whichencodes the γ-glutamyl kinase from Escherichia coli has been elucidated.The proB gene (nucleotides 259,612 to 260,715 in the sequence of GenBankaccession NC_(—)000913.2, gi: 49175990) is located between the phoE andproA genes on the chromosome of E. coli K-12.

Homoserine kinase (synonyms: B0003, ThrB, ATP:L-homoserineO-phosphotransferase) catalyzes the reaction of homoserinephosphorylation. Modification of the ThrB protein by ssrA-tagging can beuseful for L-methionine production. The activity of homoserine kinasecan be measured by the method described, for example, in Shames, S. L.and Wedler, F. C. (Arch. Biochem. Biophys., 235(2); 359-70 (1984)). Thewild-type thrB gene (synonyms: ECK0003, b0003) which encodes thehomoserine kinase from Escherichia coli has been elucidated. The thrBgene (nucleotides 2,801 to 3,733 in the sequence of GenBank accessionNC_(—)000913.2, gi: 49175990) is located between the thrA and thrC geneson the chromosome of E. coli K-12.

Homoserine dehydrogenase catalyzes the reaction of the formation ofL-aspartate-semialdehyde from homoserine. In Escherichia coli, thehomoserine dehydrogenase is the part of aspartate kinase/homoserinedehydrogenase protein encoded by thrA gene. But in other microorganisms,such as Corynebacteria, homoserine dehydrogenase is a separate enzyme.Modification of the homoserine dehydrogenase by ssrA-tagging can beuseful for L-lysine production.

The ssrA, tyrA, pheA, pgi, ilvE, ilvA, tdcB, sdaA, sdaB, argA, argG,proB, thrB, and other genes of interest can be obtained by PCR(polymerase chain reaction; refer to White, T. J. et al., Trends Genet.,5, 185 (1989)) utilizing primers prepared based on the known nucleotidesequences of the genes. Genes encoding for tag peptides from othermicroorganisms can be obtained in a similar manner.

The above-described techniques and teachings for attaching a DNAfragment able to be transcribed encoding the peptide represented in SEQID NO: 2 or a variant thereof, to the 3′-end of the genes encodingchorismate mutase/prefenate dehydrogenase and phosphoglucose isomerasecan be similarly applied to modify the activities of other proteinstranscribed from other genes of interest.

The bacterium can be obtained by introducing the aforementioned DNAsinto a bacterium which inherently have the ability to produce L-aminoacids. Alternatively, the bacterium can be obtained by imparting theability to produce L-amino acids to a bacterium which already containsthe DNAs.

L-Amino Acid-Producing Bacteria

Bacteria which are able to produce either aromatic or non-aromaticL-amino acids may be used in the present invention.

L-Phenylalanine-Producing Bacteria

Examples of parent strains for deriving L-phenylalanine-producingbacteria include, but are not limited to, strains belonging to the genusEscherichia, such as E. coli AJ12739 (tyrA::Tn10, tyrR) (VKPM B-8197);E. coli HW1089 (ATCC 55371) harboring the mutant pheA34 gene (U.S. Pat.No. 5,354,672); E. coli MWEC101-b (KR8903681); E. coli NRRL B-12141,NRRL B-12145, NRRL B-12146 and NRRL B-12147 (U.S. Pat. No. 4,407,952).Also, as a parent strain, E. coli K-12 [W3110 (tyrA)/pPHAB (FERMBP-3566), E. coli K-12 [W3110 (tyrA)/pPHAD] (FERM BP-12659), E. coliK-12 [W3110 (tyrA)/pPHATerm] (FERM BP-12662) and E. coli K-12 [W3110(tyrA)/pBR-aroG4, pACMAB] named as AJ 12604 (FERM BP-3579) may be used(EP 488424 B1). Furthermore, L-phenylalanine producing bacteriabelonging to the genus Escherichia with an enhanced activity of theprotein encoded by the yedA gene or the yddG gene may also be used (U.S.patent applications 2003/0148473 A1 and 2003/0157667 A1).

L-Histidine-Producing Bacteria

Examples of parent strains for deriving L-histidine-producing bacteriainclude, but are not limited to, strains belonging to the genusEscherichia, such as E. coli strain 24 (VKPM B-5945, RU2003677); E. colistrain 80 (VKPM B-7270, RU2119536); E. coli NRRL B-12116-B12121 (U.S.Pat. No. 4,388,405); E. coli H-9342 (FERM BP-6675) and H-9343 (FERMBP-6676) (U.S. Pat. No. 6,344,347); E. coli H-9341 (FERM BP-6674)(EP1085087); E. coli A180/pFM201 (U.S. Pat. No. 6,258,554), and thelike.

Examples of parent strains for deriving L-histidine-producing bacteriaalso include strains in which expression of one or more genes encodingan L-histidine biosynthetic enzyme are enhanced. Examples of such genesinclude genes encoding ATP phosphoribosyltransferase (hisG),phosphoribosyl AMP cyclohydrolase (hisI), phosphoribosyl-ATPpyrophosphohydrolase (hisIE), phosphoribosylformimino-5-aminoimidazolecarboxamide ribotide isomerase (hisA), amidotransferase (hisH),histidinol phosphate aminotransferase (hisC), histidinol phosphatase(hisB), histidinol dehydrogenase (hisD), and so forth.

It is known that the L-histidine biosynthetic enzymes encoded by hisGand hisBHAFI are inhibited by L-histidine, and therefore anL-histidine-producing ability can also be efficiently enhanced byintroducing a mutation conferring resistance to the feedback inhibitioninto ATP phosphoribosyltransferase (Russian Patent Nos. 2003677 and2119536).

Specific examples of strains having an L-histidine-producing abilityinclude E. coli FERM-P 5038 and 5048 which have been introduced with avector carrying a DNA encoding an L-histidine-biosynthetic enzyme (JP56-005099 A), E. coli strains introduced with rht, a gene for an aminoacid-export (EP1016710A), E. coli 80 strain imparted withsulfaguanidine, DL-1,2,4-triazole-3-alanine, and streptomycin-resistance(VKPM B-7270, Russian Patent No. 2119536), and so forth.

L-Tryptophan-Producing Bacteria

Examples of parent strains for deriving the L-tryptophan-producingbacteria include, but are not limited to, strains belonging to the genusEscherichia, such as E. coli JP4735/pMU3028 (DSM10122) and JP6015/pMU91(DSM10123) which is deficient in the tryptophanyl-tRNA synthetaseencoded by mutant trpS gene (U.S. Pat. No. 5,756,345); E. coli SV164(pGH5) having a serA allele encoding phosphoglycerate dehydrogenase freefrom feedback inhibition by serine and a trpE allele encodinganthranilate synthase not subject to feedback inhibition by tryptophan(U.S. Pat. No. 6,180,373); E. coli AGX17 (pGX44) (NRRL B-12263) andAGX6(pGX50)aroP (NRRL B-12264) deficient in the enzyme tryptophanase(U.S. Pat. No. 4,371,614); E. coli AGX17/pGX50,pACKG4-pps in which aphosphoenolpyruvate-producing ability is enhanced (WO9708333, U.S. Pat.No. 6,319,696), and the like may be used. L-tryptophan-producingbacteria belonging to the genus Escherichia with an enhanced activity ofthe identified protein encoded by and the yedA gene or the yddG gene mayalso be used (U.S. patent applications 2003/0148473 A1 and 2003/0157667A1).

Examples of parent strains for deriving the L-tryptophan-producingbacteria also include strains in which one or more activities of theenzymes selected from anthranilate synthase, phosphoglyceratedehydrogenase, and tryptophan synthase are enhanced. The anthranilatesynthase and phosphoglycerate dehydrogenase are both subject to feedbackinhibition by L-tryptophan and L-serine, so that a mutationdesensitizing the feedback inhibition may be introduced into theseenzymes. Specific examples of strains having such a mutation include aE. coli SV164 which harbors desensitized anthranilate synthase and atransformant strain obtained by introducing into the E. coli SV164 theplasmid pGH5 (WO 94/08031), which contains a mutant serA gene encodingfeedback-desensitized phosphoglycerate dehydrogenase.

Examples of parent strains for deriving the L-tryptophan-producingbacteria also include strains into which the tryptophan operon whichcontains a gene encoding desensitized anthranilate synthase has beenintroduced (JP 57-71397 A, JP 62-244382 A, U.S. Pat. No. 4,371,614).Moreover, L-tryptophan-producing ability may be imparted by enhancingexpression of a gene which encodes tryptophan synthase, among tryptophanoperons (trpBA). The tryptophan synthase consists of α and β subunitswhich are encoded by the trpA and trpB genes, respectively. In addition,L-tryptophan-producing ability may be improved by enhancing expressionof the isocitrate lyase-malate synthase operon (WO2005/103275).

L-Valine-Producing Bacteria

Example of parent strains for deriving L-valine-producing bacteria of,but are not limited to, strains which have been modified to overexpressthe ilvGMEDA operon (U.S. Pat. No. 5,998,178). It is desirable to removethe region of the ilvGMEDA operon which is required for attenuation sothat expression of the operon is not attenuated by L-valine that isproduced. Furthermore, the ilvA gene in the operon is desirablydisrupted so that threonine deaminase activity is decreased.

Examples of parent strains for deriving L-valine-producing bacteria alsoinclude mutants having a mutation of amino-acyl t-RNA synthetase (U.S.Pat. No. 5,658,766). For example, E. coli VL1970, which has a mutationin the ileS gene encoding isoleucine tRNA synthetase, can be used. E.coli VL1970 has been deposited in the Russian National Collection ofIndustrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1^(st) DorozhnyProezd, 1) on Jun. 24, 1988 under accession number VKPM B-4411.

Furthermore, mutants requiring lipoic acid for growth and/or lackingH⁺-ATPase can also be used as parent strains (WO96/06926).

L-Isoleucine-Producing Bacteria

Examples of parent strains for deriving L-isoleucine producing bacteriainclude, but are not limited to, mutants having resistance to6-dimethylaminopurine (JP 5-304969 A), mutants having resistance to anisoleucine analogue such as thiaisoleucine and isoleucine hydroxamate,and mutants additionally having resistance to DL-ethionine and/orarginine hydroxamate (JP 5-130882 A). In addition, recombinant strainstransformed with genes encoding proteins involved in L-isoleucinebiosynthesis, such as threonine deaminase and acetohydroxate synthase,can also be used as parent strains (JP 2-458 A, FR 0356739, and U.S.Pat. No. 5,998,178).

L-Leucine-Producing Bacteria

Examples of parent strains for deriving L-leucine-producing bacteriainclude, but are not limited to, strains belonging to the genusEscherichia, such as E. coli strains resistant to leucine (for example,the strain 57 (VKPM B-7386, U.S. Pat. No. 6,124,121)) or leucine analogsincluding β-2-thienylalanine, 3-hydroxyleucine, 4-azaleucine,5,5,5-trifluoroleucine (JP 62-34397 B and JP 8-70879 A); E. coli strainsobtained by the gene engineering method described in WO96/06926; E. coliH-9068 (JP 8-70879 A), and the like.

The bacterium may be improved by enhancing the expression of one or moregenes involved in L-leucine biosynthesis. Examples include genes of theleuABCD operon, which are preferably represented by a mutant leuA genecoding for isopropylmalate synthase which is not subject to feedbackinhibition by L-leucine (U.S. Pat. No. 6,403,342). In addition, thebacterium may be improved by enhancing the expression of one or moregenes coding for proteins which excrete L-amino acid from the bacterialcell. Examples of such genes include the b2682 and b2683 genes (ygaZHgenes) (EP 1239041 A2).

L-Arginine-Producing Bacteria

Examples of parent strains for deriving L-arginine-producing bacteriainclude, but are not limited to, strains belonging to the genusEscherichia, such as E. coli strain 237 (VKPM B-7925) (U.S. PatentApplication 2002/058315 A1) and its derivative strains harboring mutantN-acetylglutamate synthase (Russian Patent Application No. 2001112869),E. coli strain 382 (VKPM B-7926) (EP1170358A1), an arginine-producingstrain into which argA gene encoding N-acetylglutamate synthetase isintroduced therein (EP1170361A1), and the like.

Examples of parent strains for deriving L-arginine producing bacteriaalso include strains in which expression of one or more genes encodingan L-arginine biosynthetic enzyme are enhanced. Examples of such genesinclude genes encoding N-acetylglutamyl phosphate reductase (argC),ornithine acetyl transferase (argJ), N-acetylglutamate kinase (argB),acetylornithine transaminase (argD), ornithine carbamoyl transferase(argF), argininosuccinic acid synthetase (argG), argininosuccinic acidlyase (argH), and carbamoyl phosphate synthetase (carAB).

L-Citrulline-Producing Bacteria

Examples of parent strains for deriving L-citrulline-producing bacteriainclude, but are not limited to, strains belonging to the genusEscherichia containing mutant feedback resistant carbamoylphosphatesynthetase (U.S. Pat. No. 6,991,924) and the like.

2. Method of the Present Invention

The method of the present invention is a method for producing an L-aminoacid by cultivating the bacterium in a culture medium to produce andexcrete the L-amino acid into the medium, and collecting the L-aminoacid from the medium.

The cultivation, collection, and purification of an L-amino acid fromthe medium and the like may be performed in a manner similar toconventional fermentation methods wherein an amino acid is producedusing a bacterium.

A medium used for culture may be either a synthetic or natural medium,so long as the medium includes a carbon source and a nitrogen source andminerals and, if necessary, appropriate amounts of nutrients which thebacterium requires for growth. The carbon source may include variouscarbohydrates such as glucose and sucrose, and various organic acids.Depending on the mode of assimilation of the used microorganism,alcohol, including ethanol and glycerol, may be used. As the nitrogensource, various ammonium salts such as ammonia and ammonium sulfate,other nitrogen compounds such as amines, a natural nitrogen source suchas peptone, soybean-hydrolysate, and digested fermentative microorganismcan be used. As minerals, potassium monophosphate, magnesium sulfate,sodium chloride, ferrous sulfate, manganese sulfate, calcium chloride,and the like can be used. As vitamins, thiamine, yeast extract, and thelike, can be used.

The cultivation is preferably performed under aerobic conditions, suchas a shaking culture, and a stirring culture with aeration, at atemperature of 20 to 40° C., preferably 30 to 38° C. The pH of theculture is usually between 5 and 9, preferably between 6.5 and 7.2. ThepH of the culture can be adjusted with ammonia, calcium carbonate,various acids, various bases, and buffers. Usually, a 1 to 5-daycultivation leads to accumulation of the target L-amino acid in theliquid medium.

After cultivation, solids such as cells can be removed from the liquidmedium by centrifugation or membrane filtration, and then the L-aminoacid can be collected and purified by ion-exchange, concentration,and/or crystallization methods.

Phenylalanine produced by the method may be used for, for example,producing the lower alkyl ester of α-L-aspartyl-L-phenylalanine (alsoreferred to as “aspartame”). That is, the method includes production ofthe lower alkyl ester of α-L-aspartyl-L-phenylalanine by usingL-phenylalanine as a raw material. The method includes synthesizing thelower alkyl ester of α-L-aspartyl-L-phenylalanine from L-phenylalanineproduced by the method as described above and aspartic acid or itsderivative. As a lower alkyl ester, methyl ester, ethyl ester and propylester, or the like can be mentioned.

The process for synthesizing the lower alkyl ester ofα-L-aspartyl-L-phenylalanine from L-phenylalanine and aspartic acid orits derivative is not particularly limited and any conventional methodcan be applied so long as L-phenylalanine or its derivative can be usedfor synthesis of the lower alkyl ester of α-L-aspartyl-L-phenylalanine.Specifically, for example, the lower alkyl ester ofα-L-aspartyl-L-phenylalanine may be produced by the following process(U.S. Pat. No. 3,786,039). L-phenylalanine is esterified to obtain thelower alkyl ester of L-phenylalanine. The L-phenylalanine alkyl ester isthen reacted with L-aspartic acid derivative in which the amino groupand .beta.carboxyl group are protected, and the a-carboxyl group isesterified to activate. The derivative includes N-acyl-L-asparticanhydride such as N-formyl-, N-carbobenzoxy-, orN-p-methoxycarbobenzoxy-L-aspartic anhydride. By the condensationreaction, a mixture of N-acyl-α-L-aspartyl-L-phenylalanine andN-acyl-α-L-aspartyl-L-phenylalanine is obtained. If the condensationreaction is performed in the presence of an organic acid having an aciddissociation constant at 37° C. of 10−4 or less, the ratio of the α formto the β form in the mixture is increased (Japanese Patent Laid-OpenPublication No. 51-113841). Then the N-acyl-α-L-aspartyl-L-phenylalanineis separated from the mixture, followed by hydrogenation to obtainα-L-aspartyl-L-phenylalanine.

EXAMPLES

The present invention will be more concretely explained below withreference to the following non-limiting Examples.

Example 1 Construction of the E. coli Strain MG1655ΔtyrRtyrA-ssrA, whichContains the Mutant tyrA-ssrA Gene

1. Substitution of the Native pheA Gene and 36-nt Located at the 3′ Endof tyrA Gene Region, in E. coli with the Mutant tyrA-ssrA Gene.

To substitute the native pheA gene and 36-nt located at the 3′ end oftyrA gene region, a DNA fragment carrying 1) the 36-nt located at the 5′end of pheA gene, 2) cat gene with attL and attR sites, 3) a DNAfragment encoding the chloramphenicol resistance marker (Cm^(R)), 4) thestop codon TAA, 5) the 30-nt located at positions from 90 to 119 of thessrA gene, and 6) the 36-nt located at the 3′ end of tyrA gene wasintegrated into the chromosome of E. coli MG1655 ΔtyrR in place of thenative pheA gene and the 3′-end tyrA gene region by the method describedby Datsenko K. A. and Wanner B. L. (Proc. Natl. Acad. Sci. USA, 2000,97, 6640-6645) which is also called “Red-mediated integration” and/or“Red-driven integration” (FIG. 1).

This DNA fragment was obtained in two consecutive PCR runs where 5 μl ofamplification products from the first PCR were used as a template forthe second PCR.

The first DNA fragment containing the Cm marker encoded by the cat genewas obtained by PCR using primers P1 (SEQ ID NO: 9) and P2 (SEQ ID NO:10) and plasmid pMW118-attL-Cm-attR as a template (WO 05/010175) (FIG.2). Primer P1 contains both a region identical to the 36-nt regionlocated at the 5′ end of the pheA gene and a region complementary to the27-nt attL region of pMW118-attL-Cm-attR. Primer P2 contains both aregion identical to the 30-nt region of the ssrA gene and a regioncomplementary to the 27-nt attR region of pMW118-attL-Cm-attR. Betweenthese two regions, the stop codon TAA was inserted in primer P2.Conditions for PCR were as follows: denaturation for 5 min at 95° C.;profile for 30 cycles: 30 sec at 95° C., 30 sec at 50° C., 1 min at 72°C.; final step: 5 min at 72° C.

A 1709-bp PCR product was obtained and purified in agarose gel and 5 μlof this was used as a template for the second PCR reaction, usingprimers P1 (SEQ ID NO: 9) and P3 (SEQ ID NO: 11). Primer P3 containsboth a region complementary to the 36-nt region located at the 3′ end ofthe tyrA gene and a region identical to the 18-nt region located at the5′-end of the primer P2. Conditions for PCR were as follows:denaturation for 5 min at 95° C.; profile for 30 cycles: 30 sec at 95°C., 30 sec at 50° C., 1 min at 72° C.; final step: 5 min at 72° C.

A 1745-bp PCR product was obtained and purified in agarose gel and wasused for electroporation of the E. coli strain MG1655ΔtyrR, whichcontains the plasmid pKD46 which has a temperature-sensitive replicationorigin. The plasmid pKD46 (Datsenko, K. A. and Wanner, B. L., Proc.Natl. Acad. Sci. USA, 2000, 97:12:6640-45) includes a 2,154 nucleotideDNA fragment of phage λ (nucleotide positions 31088 to 33241, GenBankaccession no. J02459), and contains genes of the λ Red homologousrecombination system (γ, β, exo genes) under the control of thearabinose-inducible P_(araB) promoter. The plasmid pKD46 is necessaryfor integration of the PCR product into the chromosome of the E. colistrain MG1655ΔtyrR in the place of the native pheA gene and 36-ntlocated at the 3′ end of tyrA gene region.

Electrocompetent cells were prepared as follows: E. coliMG1655ΔtyrR/pKD46 was grown overnight at 30° C. in LB medium containingampicillin (100 mg/l), and the culture was diluted 100 times with 5 mlof SOB medium (Sambrook et al, “Molecular Cloning: A Laboratory Manual,Second Edition”, Cold Spring Harbor Laboratory Press, 1989) containingampicillin and L-arabinose (10 mM) [arabinose was used to induce theplasmid encoding the Red system genes]. The cells were grown withaeration at 30° C. to an OD₆₀₀ of ≈0.6 and then were madeelectrocompetent by concentrating 100-fold and washing three times withice-cold deionized H₂O. Electroporation was performed using 70 μl ofcells and 100 ng of the PCR product. The electroporation was done usinga “BioRad” electroporator (USA, No. 165-2098, version 2-89) according tothe manufacturer's instructions. The shocked cells were diluted with 1ml of SOC medium (Sambrook et al, “Molecular Cloning. A LaboratoryManual, Second Edition”, Cold Spring Harbor Laboratory Press, 1989),incubated at 37° C. for 2 h, and then spread on L-agar containingchloramphenicol (20 μg/ml) and grown at 37° C. to select Cm^(R)recombinants.

2. Verification of the pheA Gene Deletion by PCR

After 24 h of growth, colonies were tested for the presence of Cm^(R)marker by PCR using locus-specific primers P4 (SEQ ID NO: 12) and P5(SEQ ID NO: 13). For this purpose, a freshly isolated colony wassuspended in water (20 μl) and 1 μl of the obtained suspension was usedfor PCR. The temperature profile was as follows: the initial DNAdenaturation at 95° C. for 5 min; then 30 cycles (denaturation at 95° C.for 30 s, annealing at 50° C. for 30 s, and elongation at 72° C. for 1min 10 sec), and a final elongation at 72° C. for 5 min.

A few of the Cm^(R) colonies tested contained the required 2373-bp DNAfragment, confirming the substitution of the 1869-bp native region ofthe pheA gene by the hybrid tyrA-ssrA gene (see FIGS. 1 and 3). Theresulting mutant strain was named MG1655 ΔtyrR ΔpheA::cat tyrA-ssrA.

3. Substitution of the Cat Gene in E. coli MG1655 ΔtyrR ΔpheA::cattyrA-ssrA Strain with the Wild-Type pheA Gene.

To substitute the cat gene, a DNA fragment carrying the wild-type pheAgene was integrated into the chromosome of E. coli MG1655 ΔtyrRΔpheA::cat tyrA-ssrA/pKD46 in place of the cat gene by the methoddescribed by Datsenko K. A. and Wanner B. L. (Proc. Natl. Acad. Sci.USA, 2000, 97, 6640-6645) which is also called “Red-mediatedintegration” and/or “Red-driven integration”, and is also described inExample 1 (FIG. 3).

The wild-type pheA gene was obtained by PCR using the chromosomal DNA ofE. coli MG1655 as the template, and primers P4 (SEQ ID NO: 12) and P6(SEQ ID NO: 14).

Primer P6 contains both a 30-nt region identical to the region locatedat positions from 90 to 119 of the ssrA gene having the stop codon TAAand a region complementary to the 19-nt region located at the 3′ end ofthe pheA gene.

Conditions for PCR were as follows: denaturation for 5 min at 95° C.;profile for 30 cycles: 30 sec at 95° C., 30 sec at 50° C., 1 min at 72°C.; final step: 5 min at 72° C.

The amplified DNA fragment was purified by agarose gel-electrophoresis,extracted using “GenElute Spin Columns” (“Sigma”, USA) and precipitatedby ethanol. The obtained DNA fragment was used for electroporation andRed-mediated integration into the bacterial chromosome of the E. coliMG1655 ΔtyrR ΔpheA::cat tyrA-ssrA/pKD46 as described previously. Theresulting E. coli strains MG1655 ΔtyrR tyrA-ssrA/pKD46 were selected onthe minimal medium M9. The absence of L-phenylalanine in the mediumenabled the selection of colonies that were Phe⁺ with prototrophicproperties.

4. Verification of the pheA Gene Reconstruction by PCR

After 24 h of growth, colonies were tested for the presence of pheA geneby PCR using locus-specific primers P4 (SEQ ID NO: 12) and P5 (SEQ IDNO: 13). For this purpose, a freshly isolated colony was suspended inwater (20 μl) and 11 of the obtained suspension was used for PCR. Thetemperature profile was as follows: the initial DNA denaturation at 95°C. for 5 min; then 30 cycles (denaturation at 95° C. for 30 s, annealingat 50° C. for 30 s, and elongation at 72° C. for 1 min 10 sec), and afinal elongation at 72° C. for 5 min.

A few of the Phe⁺ colonies tested contained the required 1827-bp DNAfragment, confirming the substitution of the 2373-bp region of the catgene by the pheA gene (see FIG. 2). The resulting mutant strain wasnamed MG1655 ΔtyrR tyrA-ssrA/pKD46 (FIG. 4).

Then, to eliminate the pKD46 plasmid, two passages on L-agar with Cm at42° C. were performed and the colonies which appeared were tested forsensitivity to ampicillin.

Thus, a strain with the mutant tyrA gene and missing the tyrR gene wasobtained. This strain was named MG1655ΔtyrRtyrA-ssrA.

Example 2 Effect of ssrA Tagging of tyrA Gene on the L-PhenylalanineProduction

Then E. coli strains MG1655 ΔtyrR tyrA-ssrA and MG1655 ΔtyrR were eachcultivated at 37° C. for 18 hours in a nutrient broth, and 0.3 ml ofeach of these cultures was inoculated into 3 ml of fermentation mediumhaving the following composition in a 20×200 mm test tube and cultivatedat 32° C. for 72 hours with a rotary shaker.

After cultivation, the accumulated amounts of L-phenylalanine andL-tyrosine in the medium were determined by TLC. A thin-layer silica gelplate (10×15 cm) spotted with an aliquot (1-2 μl) of the culture brothwas developed with a developing solvent (2-propanol:ethylacetate:ammonia:water=16:16:3:9) and L-phenylalanine and L-tyro sinewere detected with the ninhydrin reagent. The results of four tubes offermentations are shown in Table 1.

The composition of the fermentation medium (g/l) was as follows:

Glucose 40.0 (NH₄)₂SO₄ 16.0 K₂HPO₄ 1.0 MgSO₄•7H₂O 1.0 MnSO₄•5H₂O 0.01FeSO₄•7H₂O 0.01 Yeast extract 2.0 CaCO₃ 30.0

MgSO₄.7H₂O and CaCO₃ were each sterilized separately.

It can be seen from Table 1 that MG1655 ΔtyrR tyrA-ssrA caused theaccumulation of a higher amount of L-phenylalanine and a lower amount ofL-tyrosine as compared with MG1655 ΔtyrR.

Example 3 Effect of ssrA Tagging of pgi Gene on the L-HistidineProduction

L-histidine-producing strains with the pgi gene tagged with peptides SEQID NO: 2 or SEQ ID NO: 15 were obtained. These two peptides differ bysubstitution of one of the terminal amino acid residues, either alanineor valine. These strains were obtained as follows. First, two variantsof the pgi gene containing additional nucleotide sequences at the 3′-endof the gene coding for peptides SEQ ID NO: 2 or SEQ ID NO: 15 wereobtained by PCR using chromosomal DNA of E. coli strain MG1655 andprimers pgi1 (SEQ ID NO: 16) and pgi-LAA (SEQ ID NO: 17) or pgiLVA (SEQID NO:18), respectively. Primer pgi1 is complementary to the 5′ end ofpgi gene and contains the SacI restriction site at the 5′-end thereof.Primers pgi-LVA and pgi-LAA contain regions of the 3′-end of the pgigene minus the stop-codon, a region encoding for the short peptides SEQID NO: 2 or SEQ ID NO: 15, respectively, a stop-codon, and the XbaIrestriction site at the 5′-end thereof. Second, two PCR fragments wereseparately treated with SacI and XbaI restrictases and ligated into thepMW119 plasmid which had been previously treated with the samerestrictases. The resulting plasmids were named pMW-pgi-ssrA^(LAA) andpMwpgi-ssrA^(LVA), respectively.

Then, the wild-type pgi gene was deleted in the L-histidine-producingstrain 80.

For this purpose, the DNA fragment containing the Cm^(R) marker encodedby the cat gene was obtained by PCR using primers P7 (SEQ ID NO: 19) andP8 (SEQ ID NO: 20), and plasmid pMW118-attL-Cm-attR as a template (WO05/010175) (FIG. 2). Primer P7 contains both a region identical to the36-nt region located at the 5′ end of the pgi gene and a regioncomplementary to the 28-nt attR region of pMW118-attL-Cm-attR. Primer P8contains both a region identical to the 36-nt region located at the 3′end of the pgi gene and a region complementary to the 28-nt attL regionof pMW118-attL-Cm-attR. PCR and electroporation of the obtained DNAfragment were performed as described above (see Example 1, section 1).

Deletion of the pgi gene was verified by PCR using locus-specificprimers P9 (SEQ ID NO: 21) and P10 (SEQ ID NO: 22). For this purpose, afreshly isolated colony was suspended in water (20 μl) and 1 μl of thissuspension was used for PCR. The temperature profile was as follows: theinitial DNA denaturation at 95° C. for 5 min; then 30 cycles(denaturation at 95° C. for 30 s, annealing at 50° C. for 30 s, andelongation at 72° C. for 1 min 10 sec), and a final elongation at 72° C.for 5 min.

A few Cm^(R) colonies tested contained the required 1709-bp DNA fragmentconfirming the substitution of the 1651-bp native region of the pgi geneby cat gene. The resulting mutant strain was named 80 Δpgi.

The obtained strain 80 Δpgi was transformed with the plasmidspMW-pgi-ssrA^(LAA) and pMWpgi-ssrA^(LVA) as described above. Theresulting strains were named 80 Δpgi/pMW-pgi-ssrA and 80Δpgi/pMWpgi-ssrA^(LVA), respectively.

For mini-jar batch-fermentation, one loop of each strain 80, 80Δpgi/pMW-pgi-ssrA^(LAA) and 80 Δpgi/pMWpgi-ssrA^(LVA) grown on L-agarwas transferred to L-broth and cultivated at 30° C. with rotation (140rpm) to reach an optical density of culture OD₅₄₀≈2.0. Then 25 ml ofseed culture was added to 250 ml of medium for fermentation andcultivated at 29° C. for with rotation (1500 rpm). Duration of thebatch-fermentation was approximately 35-40 hours. After the cultivationthe amount of histidine which had accumulated in the medium wasdetermined by paper chromatography. The paper was developed with amobile phase:n-butanol:acetic acid:water=4:1:1 (v/v). A solution ofninhydrin (0.5%) in acetone was used as a visualizing reagent. Theresults are shown in Table 2. It can be seen from the Table 2 thatstrains 80 Δpgi/pMW-pgi-ssrA^(LAA) and 80 Δpgi/pMWpgi-ssrA^(LVA) causedthe accumulation of a higher amount of L-histidine as compared withstrain 80.

The composition of the fermentation medium (pH 6.0) (g/l):

Glucose 50.0 Mameno 0.2 of TN (NH₄)₂SO₄ 8.0 KH₂PO₄ 0.5 MgSO₄ 7H₂0 0.4FeSO₄ 7H₂0 0.02 MnSO₄ 0.02 Thiamine 0.001 Betaine 2.0 L-proline 0.8L-glutamate 3.0 L-aspartate 1.0 Adenosine 0.2

While the invention has been described in detail with reference topreferred embodiments thereof, it will be apparent to one skilled in theart that various changes can be made, and equivalents employed, withoutdeparting from the scope of the invention. All the cited referencesherein are incorporated as a part of this application by reference.

TABLE 1 Strain OD₅₄₀ Phe, g/l Tyr, g/l MG1655ΔtyrR 28.5 ± 0.6 0.13 ±0.01 0.017 ± 0.002 MG1655ΔtyrR tyrA-ssrA 26.3 ± 0.4 0.46 ± 0.04 traces

TABLE 2 Amount of Yield per Strain OD₄₅₀ histidine, g/l glucose, % 80(VKPM B-7270) 31.0 9.0 18.0 80 Δpgi 20.2 8.1 16.2 80Δpgi/pMW-pgi-ssrA^(LAA) 23.2 10.7 21.4 80Δpgi/pMW-pgi-ssrA^(LVA) 22.810.6 21.2

1. An L-amino acid producing bacterium of the Enterobacteriaceae familycomprising a DNA comprising: A) a gene encoding a bacterial enzyme whichinfluences L-amino acid biosynthesis, and B) a DNA fragment able to betranscribed and encoding the peptide of SEQ ID NO: 2, or a variantthereof, wherein said fragment is attached to said gene at the 3′ end ofsaid gene.
 2. The bacterium according to claim 1, wherein said bacteriumbelongs to the genus Escherichia.
 3. The bacterium according to claim 1,wherein said bacterium is an L-phenylalanine producing bacterium.
 4. Thebacterium according to claim 1, wherein the enzyme is chorismatemutase/prephenate dehydrogenase.
 5. The bacterium according to claim 1,wherein said bacterium is an L-histidine-producing bacterium.
 6. Thebacterium according to claim 1, wherein the enzyme is phosphoglucoseisomerase.
 7. A method for producing an L-amino acid comprisingcultivating the bacterium according to claim 1 in a culture medium, andisolating the L-amino acid from the culture medium.
 8. The methodaccording to claim 7, wherein said L-amino acid is L-phenylalanine. 9.The method according to claim 7, wherein said L-amino acid isL-histidine.
 10. A method for producing a lower alkyl ester ofα-L-aspartyl-L-phenylalanine, comprising cultivating the bacteriumaccording to claim 1 in a culture medium to produce and accumulateL-phenylalanine in the medium, and synthesizing the lower alkyl ester ofα-L-aspartyl-L-phenylalanine from aspartic acid or a derivative thereofand the obtained L-phenylalanine.
 11. The method according to claim 10,further comprising esterifying L-phenylalanine to generate a lower alkylester of L-phenylalanine, condensing the lower alkyl ester ofL-phenylalanine with the aspartic acid derivative, wherein thederivative is N-acyl-L-aspartic anhydride, separating the lower alkylester of N-acyl-α-L-aspartyl-L-phenylalanine from the reaction mixture,and hydrogenating the lower alkyl ester ofN-acyl-α-L-aspartyl-L-phenylalanine to generate the lower alkyl ester ofα-L-aspartyl-L-phenylalanine.